J. Embryol. exp. Morph. 88, 231-247 (1985) 231 Printed in Great Britain © The Company of Biologists Limited 1985 The ontogenesis of cranial neuromeres in the rat embryo II. A transmission electron microscope study FIONA TUCKETT AND GILLIAN M. MORRISS-KAY Department of Human Anatomy, University of Oxford, South Parks Road, Oxford OX1 3QX, U.K. SUMMARY The morphogenesis of rhombomeres (neuromeres) caudal to the preotic sulcus during neurulation in rat embryos is described. A model is proposed to explain the development of the characteristic neuromeric sulci and interneuromeric gyri based on the cytoskeletal elements and the kinetic behaviour of the neural epithelium. Evidence obtained from a study of control, cytochalasin D-treated and colchicine-treated embryos, at the electron microscopic level, supports the proposed model. The longitudinally expanding cranial neural epithelium bulges between microtubule blocks present within the interneuromeric gyri, causing a bulge to develop along the line of least resistance, away from the microfilament-rich luminal border of the neuromeric sulcus region. INTRODUCTION Neuromeres have been observed as a segmental arrangement of sulci and gyri within the early neural tube of all vertebrate embryos. Their characteristic morphology is illustrated in Fig. 1. This study addresses the question of how the sulci and gyri develop from an initially straight neural epithelium, and how their structure is maintained. Observations on a variety of vertebrates (chick, fish, urodeles) led Kallen (1956) to propose that the formation of neuromeric sulci was the result of mitotic patterning within the neural epithelium. His model is based on transverse sections of the neural epithelium during the formation of a neuromere, which he termed a 'proliferation centre'. According to Kallen, the furrowing of the luminal border results from the redistribution of neuroepithelial cells, due to the mitotic patterning, while the bulging at the basal border is brought about by the formation of proportionally more cells in the centre of the region, where the mitotic activity is highest. This interpretation relies strictly on the kinetics of the cells within the neuromere and their movement across the epithelium between the basal and luminal borders, and takes no account of the possible migration of cells within the Key words: rat embryo, neuromeres, neural tube, cytochalasin D, colchicine, cranial neuromeres. 232 F. TUCKETT AND G. M. MORRISS-KAY neural epithelium (along the length of the embryo), or of possible regulation of shape through a periodicity of structural components. We have suggested that a localized increase in the mitotic index is the initial means of identifying a neuromere, and once the neuromere is expressed morphologically as a surface sulcus the mitotic index returns to its basal level (Tuckett, 1984; Tuckett, Lim & Morriss-Kay, 1985). Kallen's 'proliferation centre' appears to refer to the localized concentrations of mitotic activity which we have observed in both transverse and coronal section. This study investigates the possibility of an alternative hypothesis for the development of neuromeres which embraces both the mitotic patterning (orientation of the mitotic spindle axes) and the cytoskeletal components of the neural epithelium. This hypothesis is illustrated diagrammatically in Fig. 2. It proposes that growth of the neural tube is generated longitudinally by cell division in this plane, but that elongation is prevented by the fixed nature of the tube within the embryo. In consequence, the lengthening epithelium bulges outwards Fig. 1. A light micrograph of a coronal section, along the neural tube of a 16-somite- stage embryo, at the level of the otocysts (o). The lumen of the neural tube is narrowed caudally (to the right). The neural epithelium (nep) is folded into a series of sulci and gyri which is characteristic of neuromeres. The centre of each neuromere, the sulcus, is arrowed; mitotic figures with their darkly staining chromatin are generally localized within the sulci. The interneuromeric junctions (gyri) are marked with asterisks (*). Cranial mesenchyme (mes) underlies the neural epithelium. Alcian-blue-stained wax section (5/mi) from Tuckett (1984). Scale bar represents 40/mi. Ontogenesis of cranial neuromeres studied by TEM 233 ABC • nep mes mes m * b gyrus gyrus t t 1™ sulcus sulcus • • b • • Fig. 2. Schematic diagram representing coronal sections through a similar part of the myelencephalon to that shown in Fig. 1. b, 'block'; nep, neural epithelium; mes, mesenchyme; m, microfilaments. (A) Before generation of a neuromere. Growth of the neural tube is generated by cell division within the longitudinal plane; however the neural epithelium is fixed at various points along the tube, thus forming 'blocks' to tube elongation. The luminal border is rich in microfilament bundles which contract along the line indicated by the arrows. (B) After generation of a neuromere. The lengthening epithelium has to bulge either outward, or inward along the line of least resistance. The microfilament-bound luminal border prevents inward bulging of the neural epithelium, and thus an outward bulge into the more easily deformable mesenchyme results. Due to the structural integrity of the neural epithelium and as a consequence of the outward bulging, a sulcus develops at the luminal border. The blocks to tube elongation are localized at the interneuromeric junctions (gyri). (C) Modification of the model following the studies reported in this paper. The gyrus region consists of a fan-shaped arrangement of cells with microtubule groups aligned perpendicular to the luminal border. into the adjacent mesenchyme since the basal border (lacking in microfilaments) is more easily deformable than the microfilament-bound luminal border. The repeating pattern of sulci and gyri results from the presence of a series of blocks to deformation (gyri) between the bulging regions (sulci). This hypothesis is based on evidence from a previous study (Tuckett & Morriss-Kay, 1985), as follows: (i) cell division within the neural epithelium at relevant stages is predominantly (98%) orientated with the mitotic spindle axis parallel to the long axis of the embryo, so as to increase the length of the neural epithelium; (ii) rostral to the preotic sulcus cells generated in the midbrain and upper hindbrain appear to flow rostrally to provide an extrinsic source of cells for the rapidly expanding forebrain, suggesting the existence of a block to cell movement at or close to the preotic sulcus. Since we are now suggesting a series of blocks to cell movement between neuromeric sulci, the present hypothesis relates only to the clearly segmented caudal metencephalon and myelencephalon, i.e. the region caudal to the preotic sulcus. 234 F. TUCKETT AND G. M. MORRISS-KAY To examine the feasibility of this model for the development of neuromere morphology, and to investigate the morphogenetic nature of the proposed blocks to cell movement between neuromeres, transmission electron microscopy was employed. The function of microtubules and microfilaments in maintaining the morphology was examined by treating embryos in vitro with colchicine which binds to tubulin and prevents microtubule assembly, and cytochalasin D which inhibits microfilament assembly; each may also disrupt existing microtubules and micro- filaments respectively. MATERIALS AND METHODS Wistar strain rat embryos were explanted in Tyrode's saline on day 10 of pregnancy (day of positive vaginal smear = day 0). A total of 39 embryos was used. In the 34 embryos which were subsequently to be cvltured only Reichert's membrane was opened; in the remaining 5 embryos the extraembryonic membranes were removed before fixation. At the time of fixation the embryos had between 12 and 16 somite pairs. Whole embryo culture Embryos were cultured at a temperature of 38 °C in 60 ml Pyrex glass bottles containing 2-5 ml immediately centrifuged, heat-inactivated rat serum (Steele & New, 1974), 2-5 ml Tyrode's saline and lOjul penicillin/streptomycin (5000 i.u. ml"1 and SOOOjUgmP1). The bottles were gassed with 5 % CO2:5 % O2:90 % N2 (New, Coppola & Cockroft, 1976a,b) prior to sealing. The bottles were continuously rotated at 30r.p.m. At the end of the culture period, the embryos were thoroughly washed in Tyrode's saline and the extraembryonic membranes were removed before fixation. Cytochalasin D-treated embryos A stock solution of l-Omgml"1 cytochalasin D (Sigma, London) was prepared in a 10% solution of dimethylsulphoxide (DMSO; Sigma, London) and stored at -20°C. 7-5/il of the stock solution was added to the culture medium (final concentration of 0-15 fig ml"1). Ten embryos at the 11- to 13-somite stage were cultured in the presence of cytochalasin D for 2h. Five control embryos were cultured in the presence of 7 • 5 /A of a 10 % solution of DMSO for 2 h. Colchicine-treated embryos 14 embryos at the 11- to 13-somite stage were treated in culture for 3 h with colchicine (Sigma, London) which had been added to the culture medium so as to produce a final concentration of 0-2 jug ml"1. This concentration was found to cause a sufficiently high mitotic arrest without adversely affecting the gross morphology of the embryo; the duration of colchicine treatment was less than half the cell-cycle time which we have determined previously (Tuckett & Morriss- Kay, 1985), and thus only a small number of cells were observed to be arrested at the metaphase stage. Subsequent treatment was the same as for the untreated, control embryos. Five embryos were cultured in unsupplemented culture medium for 3 h. Electron microscopy After removal of the membranes, embryos were fixed in 2-5 % cacodylate-buffered osmium tetroxide, washed and dehydrated through graded alcohols before embedding in Spurr resin. Thin sections of 80-90 nm thickness were collected on copper grids and double stained with uranyl acetate at 40°C for 45 min and lead citrate for 5 min at room temperature. Ontogenesis of cranial neuromeres studied by TEM 235 Light microscopy Semi thin sections of 1 /an thickness were cut immediately adjacent to the thin sections.